This is a competitive renewal application to further characterize the molecular and cellular facets of host immunity in high-risk corneal transplants, performed in inflamed host beds and distinguished by their rapid rejection. The goal of our grant, which has been supported since 2000, is to gain new insights into the mechanisms that distinguish high-risk vs. low-risk transplant immunity. Our work has identified Foxp3hi regulatory T cell (Treg) function as being critical for allotolerance and graft survival. Our overarching hypothesis is that the high-risk graft is characterized by a microenvironment which abrogates the tolerogenic potential of Tregs, rendering them incapable of protecting the allograft against immune- mediated attack. This is supported by our data from transgenic mice demonstrating that in high-risk grafted hosts some Tregs lose Foxp3 expression and convert into `exTreg' that express interferon-gamma (IFN?), an inflammatory cytokine which can exert damage on graft tissue. We thus propose to pursue the following three specific aims: In Aim 1, we will determine the function of Tregs in protecting graft endothelial cells against effector T cell-mediated (Aim 1A) and inflammatory cytokine-mediated (Aim 1B) attack. In Aim 2 we plan to explore the function of different Treg phenotypes in regulating corneal angiogenesis, an entirely novel area. Surprisingly, we have found that Foxp3-/lo Tregs, which have limited immunosuppressive function, are potently anti-angiogenic. We will quantify the expression of different VEGF species and the anti-angiogenic cytokine IFN? by Tregs derived from low-risk and high-risk grafted hosts, hypothesizing that a high IFN?/VEGF ratio determines Treg angiostatic function. In Aim 3 we test the hypothesis that exTregs not only have defective immunoregulatory function, but actually effect graft rejection, a wholly novel concept. In Aim 3A we will determine the capacity of exTreg to induce graft loss, and in Aim 3B we will determine the cytokine cues that induce Treg conversion to exTregs, devising strategies for preventing this conversion and thus promoting graft tolerance. Our study design relies on using the core expertise of our laboratory along with use of well-characterized mouse models of corneal transplantation in conjunction with in vitro immunological and cell proliferation assays and use of a double transgenic mouse that permits us to monitor the differentiation of Tregs to exTregs. The overall health relevance of this research is that corneal grafting represents the number one form of transplantation performed in the United States. However, while most high-risk corneal transplant patients rapidly reject their grafts, there has been no significant change in the management of high-risk transplants for decades. Thus, the long-term objective of our project is to use data derived from our studies to develop new strategies to promote graft acceptance without the use of systemic immunosuppressive regimens that place grafted recipients at significant risk of serious morbidities.